Tyrosine hydroxylase (TH), in the presence of a cofactor synthesized and regenerated by dihydropteridine reductase (DHPR), catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in catecholamine neurotransmitter synthesis. The regulation of TH, and the role played by DHPR in regenerating tetrahydrobiopterin (BH4), the obligatory cofactor for TH, represent critical processes in neurotransmission. Although BH4 acts as a cofactor in the hydroxylation of aromatic amino acids, it can also serve as a negative effector by inactivating or destabilizing the hydroxylases. The milecular basis of this dual role is presently unknown. Our proposed biochemical characterization of TH and DHPR should shed new liht on how these enzymes function in neurotransmitter synthesis. The proposed studies will elucidate the protein structure and sequence of functionally significant fragments of TH and DHPR. These enzymes will be purified from rat tissues by conventional procedures, including salt fractionation, ion exchange and affinity chromatography and HPLC. Monoclonal antibodies produced against these enzymes will be employed as reagents for specific affinity chromatography purification methods. Those of appropriate specificity will be used as probes to characterize the phosphorylation and BH4 binding sites in TH, and the NADH binding sites in DHPR, using enzyme obtained from rat adrenal glands and striatal synaptosomes.